title: Glutamine depletion induces murine neonatal melena with increased apoptosis of the intestinal epithelium.
abstract: OBJECTIVE
  To investigate the possible biological outcome and effect of glutamine depletion in neonatal mice and rodent intestinal epithelial cells.
  
  
  METHODS
  We developed three kinds of artificial milk with different amounts of glutamine; Complete amino acid milk (CAM), which is based on maternal mouse milk, glutamine-depleted milk (GDM), and glutamine-rich milk (GRM). GRM contains three-fold more glutamine than CAM. Eighty-seven newborn mice were divided into three groups and were fed with either of CAM, GDM, or GRM via a recently improved nipple-bottle system for seven days. After the feeding period, the mice were subjected to macroscopic and microscopic observations by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and Ki-67 as markers of cell proliferation, and for cleaved-caspase-3 as a marker of apoptosis. Moreover, IEC6 rat intestinal epithelial cells were cultured in different concentrations of glutamine and were subject to a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate cell proliferation assay, flow cytometry, and western blotting to examine the biological effect of glutamine on cell growth and apoptosis.
  
  
  RESULTS
  During the feeding period, we found colonic hemorrhage in six of 28 GDM-fed mice (21.4%), but not in the GRM-fed mice, with no differences in body weight gain between each group. Microscopic examination showed destruction of microvilli and the disappearance of glycocalyx of the intestinal wall in the colon epithelial tissues taken from GDM-fed mice. Intake of GDM reduced BrdU incorporation (the average percentage of BrdU-positive staining; GRM: 13.8%, CAM: 10.7%, GDM: 1.14%, GRM vs GDM: P < 0.001, CAM vs GDM: P < 0.001) and Ki-67 labeling index (the average percentage of Ki-67-positive staining; GRM: 24.5%, CAM: 22.4% GDM: 19.4%, GRM vs GDM: P = 0.001, CAM vs GDM: P = 0.049), suggesting that glutamine depletion inhibited cell proliferation of intestinal epithelial cells. Glutamine deprivation further caused the deformation of the nuclear membrane and the plasma membrane, accompanied by chromatin degeneration and an absence of fat droplets from the colonic epithelia, indicating that the cells underwent apoptosis. Moreover, immunohistochemical analysis revealed the appearance of cleaved caspase-3 in colonic epithelial cells of GDM-fed mice. Finally, when IEC6 rat intestinal epithelial cells were cultured without glutamine, cell proliferation was significantly suppressed after 24 h (relative cell growth; 4 mmol/L: 100.0% ± 36.1%, 0 mmol/L: 25.3% ± 25.0%, P < 0.05), with severe cellular damage. The cells underwent apoptosis, accompanied by increased cell population in sub-G0 phase (4 mmol/L: 1.68%, 0.4 mmol/L: 1.35%, 0 mmol/L: 5.21%), where dying cells are supposed to accumulate.
  
  
  CONCLUSIONS
  Glutamine is an important alimentary component for the maintenance of intestinal mucosa. Glutamine deprivation can cause instability of the intestinal epithelial alignment by increased apoptosis.
authors.affiliation: Department of Gastroenterological Surgery, Transplant, and Surgical Oncology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1, Shikata-cho, Okayama, 700-8558, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Motoki
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Naomoto
authors.forename: Junji
authors.initials: J
authors.lastname: Hoshiba
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shirakawa
authors.forename: Tomoki
authors.initials: T
authors.lastname: Yamatsuji
authors.forename: Junji
authors.initials: J
authors.lastname: Matsuoka
authors.forename: Munenori
authors.initials: M
authors.lastname: Takaoka
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Tomono
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsuchita
authors.forename: Mehmet
authors.initials: M
authors.lastname: Gunduz
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nagatsuka
authors.forename: Noriaki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Toshiyoshi
authors.initials: T
authors.lastname: Fujiwara
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D003106:Colon / Q000473:pathology; D004032:Diet; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D005973:Glutamine / Q000008:administration & dosage / Q000172:deficiency*; D006470:Hemorrhage; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000473:pathology*; D008551:Melena / Q000378:metabolism*; D051379:Mice; D011247:Pregnancy; D051381:Rats
chemical_list: D005973:Glutamine